Polyvinyl difluoride (PVDF) membranes are commonly used as a solid support in a variety of immunological assays. Due to its high capacity to bind and retain protein, PVDF is an ideal membrane particularly for assays, such as the ELISpot and microarray assays, that are dependent on a high local concentration of the immobilized agent. However, to achieve its high binding properties the PVDF needs to be activated since the membrane in its original form has very hydrophobic properties and comparatively poor binding properties. Activation is normally achieved by treatment with ethanol or methanol which makes the membrane more hydrophilic allowing for aqueous solutions to penetrate into the membrane and thereby increasing the surface area accessible for binding. Typically, the membrane is treated with ethanol for a short period, followed by several rounds of washing before addition of the substance to be coated. It is essential that the membrane is not allowed to dry between the activation and the coating as it then resumes its hydrophobic low-binding properties.
The activation procedure needs to be performed in a strictly defined manner, particularly when the membrane is used for cellular assays, since “overtreatment” (i.e. too much ethanol or too long a treatment duration) has been shown to have a deleterious effect and lead to suboptimal results.